Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics

Anna M L Coenen-Stass,Marie J Pauwels,Britt Hanson,Carla Martin Perez,Mariana Conceição,Matthew J A Wood,Imre Mäger,Thomas C Roberts

doi:10.1080/15476286.2019.1582956

Anna M L Coenen-Stass, Marie J Pauwels + Show 6 more

Open Access

https://doi.org/10.1080/15476286.2019.1582956

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Abstract

ABSTRACT Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4ºC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37ºC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from ~1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells.

Highlights

Extracellular microRNAs are present in a wide variety of biofluids, including serum, plasma, urine, cerebral spinal fluid, saliva, tears, milk, and seminal fluid [1]
To investigate the stability of ex-miRNAs, we sought to determine the half-lives of myomiRs and non-myomiR controls at physiological temperature (i.e. 37°C) in conditioned medium taken from C2C12 myotube cultures
C2C12 cells were cultured in differentiation conditions for 6 days, so as to reach a stage at which ex-myomiRs were being secreted into the cell culture medium at high levels [14]

Summary

Introduction

Extracellular microRNAs (ex-miRNAs) are present in a wide variety of biofluids, including serum, plasma, urine, cerebral spinal fluid, saliva, tears, milk, and seminal fluid [1]. A set of highly muscle-enriched miRNAs, the classical myomiRs (consisting of the miR-1/206 family, MIPF0000038, and miR-133 family, MIPF0000029 [18]) are elevated in the serum of DMD patients and dystrophic animal models [10,11,12,13,14,15,16,17]. These myomiRs are known to regulate myoblast proliferation and differentiation by suppressing expression of stemness factors such as PAX3 and PAX7, inhibiting DNA synthesis via POLA1, and promoting MEF2 expression via the repression of HDAC4 [19,20,21,22]. Whether or not these ex-miRNAs can act to transfer gene regulatory information between cells remains an exciting area of ongoing research [26,27,28]

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